Progression Of Oral Premalignant Lesions Research Articles

A Multi-Modal Risk Score to Predict Progression of Oral Premalignant Lesions to Oral Squamous Cell Carcinoma

A Multi-Modal Risk Score to Predict Progression of Oral Premalignant Lesions to Oral Squamous Cell Carcinoma

Local Anti-PD-1 Delivery Prevents Progression of Premalignant Lesions in a 4NQO-Oral Carcinogenesis Mouse Model.

Although the principle of systemic treatment to prevent the progression of oral premalignant lesions (OPL) has been demonstrated, there remains a lack of consensus about an optimal approach that balances clinical efficacy with toxicity concerns. Recent advances in cancer therapy using approaches targeting the tumor immune microenvironment (TIME) including immune-checkpoint inhibitors indicate that these agents have significant clinically activity against different types of cancers, including oral cancer, and therefore they may provide an effective oral cancer prevention strategy for patients with OPLs. Our past work showed that systemic delivery of a monoclonal antibody to the programmed death receptor 1 (PD-1) immune checkpoint can inhibit the progression of OPLs to oral cancer in a syngeneic murine oral carcinogenesis model. Here we report a novel approach of local delivery of a PD-1 immune-checkpoint inhibitor loaded using a hydrogel, which significantly reduces the progression of OPLs to carcinomas. In addition, we detected a significant infiltration of regulatory T cells associated with oral lesions with p53 mutation, and a severe loss of expression of STING, which correlated with a decreased infiltration of dendritic cells in the oral lesions. However, a single local dose of PD-1 inhibitor was found to restore stimulator of interferon response cGAMP interactor 1 (STING) and CD11c expression and increase the infiltration of CD8+ T cells into the TIME irrespective of the p53 mutational status. Overall, we provide evidence for the potential clinical value of local delivery of biomaterials loaded with anti-PD-1 antibodies to prevent malignant progression of OPLs. PREVENTION RELEVANCE: Oral cancer is an aggressive disease, with an overall survival rate of 50%. Preinvasive histologic abnormalities such as tongue dysplasia represent an early stage of oral cancer; however, there are no treatments to prevent oral carcinoma progression. Here, we combined biomaterials loaded with an immunotherapeutic agent preventing oral cancer progression.

Targeting of CD40 and PD-L1 Pathways Inhibits Progression of Oral Premalignant Lesions in a Carcinogen-induced Model of Oral Squamous Cell Carcinoma.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1-knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting. PREVENTION RELEVANCE: PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

Abstract PO048: CD40/anti-PD-1 sequential immunotherapy outperforms multiple immunotherapy combinations in an oral cancer prevention mouse model

Abstract PD-1 checkpoint inhibitors pembrolizumab and nivolumab are approved for the treatment of recurrent or metastatic oral squamous cell carcinoma (OSCC). Previously we have demonstrated that PD-1/PD-L1 pathway blockade can reduce the progression of oral premalignant lesions (OPL) to carcinoma in the 4-NQO carcinogen-induced murine model of OSCC. Furthermore, pembrolizumab is currently being evaluated for prevention of OSCC in patients with high risk OPLs in the Immune Prevention of Oral Cancer clinical trial (NCT02882282). Building on these earlier studies, here we have conducted pre-clinical evaluation of other possible targets including CD40, OX40, and 4-1BB for the immunoprevention of OSCC, and determined the impact of combined or sequential treatment with PD-1/PD-L1 pathway blockade. We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO, mice were treated with either IgG (n: 20), used as control group, anti-PD-1 monotherapy, concomitant anti-PD-1+OX4, anti-PD-1+CD40, anti-PD-1+4-1BB, or OX40, CD40, and 4-1BB followed by anti-PD-1 therapy (n:10 treatment groups). Mice were sacrificed 8 weeks after treatment. We assessed serial H&E-stained cross sections of tongues harvested at the same time point for total OSCC area. Tumor draining lymph nodes (TDLN) lymphocytes were analyzed by flow cytometry. Anova and Mann-Whitney test were used for statistical comparison. Treatment of mice harboring OPLs with CD40 followed by anti-PD-1 immunotherapy resulted in a decrease in the median total OSCC area when compared with our IgG control group (p= 0.034). Treatment with anti-PD-1 monotherapy showed a trend towards reducing progression of OPL to OSCC. Concomitant therapy with CD40 and anti-PD-1 along with the other combination immunotherapy regimens did not demonstrate any significant reduction in the progression of OPL to OSCC. Treatment with sequential CD40+anti-PD-1 increased both the CD8+PD-1+ lymphocyte population and the MFI values for PD-1 in the CD4+Foxp3- population in TDLN. When the TDLN of both concomitant and sequential 4-1BB+anti-PD-1 treatment groups were analyzed, a decrease in the CD4+PD-L1+ population, and an increase in both CD8+PD-1+ population and CD8/CD4+PD-L1+ ratio was found, although this change in the immune milieu was not associated to a reduction of OPL progression to OSCC. Targeting CD40 and PD-1 pathways sequentially, but not concomitantly, during the OPL stage led to a reduction in the progression of OPL to OSCC. The TDLN analysis of this treatment group revealed an increase in both CD8+PD-1+ lymphocytes and MFI values for PD-1 in CD4+Foxp3- lymphocytes. No other treatment group was able to significantly reduce progression of OPL to OSCC or showed a similar modulation of the lymphocyte population. These results support further investigation on how sequential CD40+anti-PD-1 immunotherapy hinder tumor immune evasion, making it a potential regimen for prevention of OSCC. Citation Format: Jose A. Monteiro de Oliveira Novaes, Alissa Poteete, Marlese Pisegna, William N. William, John V. Heymach. CD40/anti-PD-1 sequential immunotherapy outperforms multiple immunotherapy combinations in an oral cancer prevention mouse model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO048.

Abstract 2548: Pentose phosphate pathway inhibition potentiates metformin cytotoxic effects on HNSCC cells

Abstract The pentose phosphate pathway (PPP) enables cancer cells to adapt to anabolic demands that require rapid DNA, RNA, and lipid synthesis and to oxidative cellular stress. Metformin can efficiently inhibit the malignant progression of oral premalignant lesions by indirectly inhibiting the mammalian target of rapamycin (mTORC1). Frequently activated in cancer cells, mTORC1 may upregulate the oxidative branch of the pentose phosphate pathway (PPP) through the sterol regulatory element binding protein (SREBP1). Our previous results showed that the combination of metformin and 6-AN (a PPP inhibitor) strongly potentiates their individual cytotoxic effects on melanoma, glioblastoma, mammary and colon carcinoma cell lines. However, the mechanism of this synergism remains unclear. In this context, the aim of this project was to determine the cell signaling events underlying the therapeutic potential of this combinatory approach, against HNSCC cell lines. Then, we study the effect of metformin alone or in combination with 6-AN on AMPK/mTOR/S6K/S6 and SREBP1 pathway. We found that the inhibition of PPP strongly potentiated metformin cytotoxic effect on 2D and 3D cultures of CAL33 and HN12, both HNSCC cell lines. Furthermore, the combination synergized to increase the activation of AMPK, concomitant with decreased phosphorylation of mTOR, decreased phosphorylation of S6K and decreased phosphorylation of S6. Interestingly, activation of SREBP1 was also synergistically decreased by this combinatory approach. We conclude that the inhibition of PPP and metformin converges to inhibit mTOR pathway. This interaction deserves to be further investigated as a potential strategy to potentiate metformin therapy. Citation Format: Marcela Solange Villaverde, Jorge Silvio Gutkind. Pentose phosphate pathway inhibition potentiates metformin cytotoxic effects on HNSCC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2548.

Effect of 5-ALA-mediated photodynamic therapy on mast cell and microvessels densities present in oral premalignant lesions induced in rats

Acute inflammatory response after photodynamic therapy is frequently described, and increase on mast cell degranulation is also present during this process. The mast cell activation may improve angiogenesis, and this fact has been associated with progression of oral premalignant lesions (OPL). The aim of this study was to evaluate whether photodynamic therapy (PDT) increases mast cell density (MCD) and microvessels density (MVD) in 4-nitroquinoline-1-oxide(4NQO)-induced OPL in rats. 4NQO-induced OPL were treated or not with 5-ALA followed by laser irradiation (PDT group and 4NQO groups, respectively). Mast cells and CD34+ microvessels were counted. Both PDT and 4NQO groups had MCD and MVD that were higher than normal mucosa (p<0.05). The 4NQO group had the lowest number of non-degranulated MCD in comparison to experimental periods of PDT (PDT 6h – p=0.020; 24h – p=0.016; 48h – p=0.003; 72h – p=0.033). Only in the PDT group did MCD and MVD have a significant correlation (r=0.6219, p=0.010). 5-ALA-mediated PDT modified the MCD and MVD in the induced OPL, leading to degranulation of mast cells and angiogenesis. A PDT protocol with an efficient eradication of the OPL must be adopted considering the angiogenesis potential associated with the mast cell activation after the therapy.

In Vivo Antineoplastic Effects of the NSAID Sulindac in an Oral Carcinogenesis Model.

The antineoplastic properties of the NSAID sulindac have long been studied. The purpose of this study was to explore sulindac's in vivo effects on oral squamous cell carcinoma (SCC) oncogenesis using the hamster cheek pouch oral carcinogenesis model (HOCM). Thirty Syrian golden hamsters were divided into three experimental and two control groups (n = 6 each). The animals' right buccal pouches were treated with carcinogen for 9 weeks in one experimental and one control group and for 14 weeks in all other three groups. The animals of two experimental groups received sulindac from the 1st week and those of the third experimental group from the 10th week. After the end of carcinogenesis, treated buccal pouches were removed and examined. In animals treated with carcinogen for 14 weeks, development of oral SCC and tumor volume were significantly lower in animals that received sulindac from the first week of the experiment. Oral SCC developing in animals that received sulindac were more frequently well differentiated compared with the control group. In animals treated with carcinogen for 9 weeks, the animals that received sulindac developed lower grade of epithelial dysplasia. Proliferation index Ki-67 and positivity for the antiapoptotic molecule survivin were lower in the animals that received sulindac. Treatment with sulindac appears to delays the progression of oral premalignant lesions to oral SCC in the HOCM, also resulting in smaller and better differentiated tumors. These in vivo antineoplastic effects may be related to sulindac's ability to decrease cell proliferation and to prevent survivin expression.

Abstract 2269: Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2

Abstract Purpose: Effective chemopreventive strategies could save millions of lives. We have previously reported that the combination of the EGFR-TKI erlotinib and the green tea constituent epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. Methods: Well characterized and genetically validated squamous cell carcinoma of the head and neck cell lines were used throughout the study. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Gene overexpression and knockdown strategies were used to activate or shut down the expression of specific proteins. Results: siRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p=). On the other hand, overexpression of Bcl-2 significantly protected cells from apoptosis (p=), whereas overexpression of constitutively AKT had minimal effect on apoptosis (actual values). Analysis of mRNA expression by RT-PCR revealed that neither erlotinib, EGCG nor their combination had any significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2, suggesting post-transcriptional regulation of Bim, p21, p27 and Bcl-2 by the combination of erlotinib and EGCG. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim, and that inhibition of protein translation by cycloheximide pretreatment stabilized Bim, suggesting posttranslational regulation of Bim. Conclusion: Our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the post-translational level. Currently, a clinical trial is underway at Winship Cancer Institute of Emory University to inhibit or reverse the progression of oral premalignant lesions using this combination. (This study is supported by R03CA159369, P50CA128613 and Robbins Scholar Award of Winship Cancer Institute). Citation Format: Abedul Haque, Mohammad A. Rahman, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2269. doi:10.1158/1538-7445.AM2014-2269

Abstract CN14-04: Identifying the premalignant lesion at high risk for progression to cancer

Abstract Oral tumorigenesis is a multistep process requiring the accumulation of multiple molecular alterations such as mutations, genomic deletions and amplifications, as well as epigenetic modifications in oral epithelial cells. These alterations allow the affected cells gaining growth advantages over other cells and other malignant phenotypes. In the past two decades, extensive studies have been conducted to understand molecular abnormalities in oral cancers and oral premalignant lesions (OPLs). Although promising, none of these has translated into a clinical test which can improve patient classification or cancer risk assessment. OPLs can exhibit a variety of histological features from hyperkeratosis, hyperplasia, and mild dysplasia to severe dysplasia. Together with other clinical features, our current practice has limited capability to accurately stratify cancer risk of patients with OPL. Such stratification is critical in our effort to develop surveillance measures for early cancer diagnosis and interventions to prevent or delay cancer development. In this presentation, I will focus on a series of biomarker discovery studies based on patients with OPL and enrolled in a prospective chemoprevention trial to illustrate some of the current efforts in oral cancer risk assessment based underlying molecular abnormalities in OPLs. The chemoprevention trial randomized 167 patients with biopsies obtained at the time of enrollment and certain time points during follow-up. One hundred and sixty-two patients had oral cancer-free survival data with the median time of follow-up about 7.5 years. During the follow-up period, 38 patients developed oral cancer but no statistically significant difference was observed in any of the treatment arms. We examined podoplanin expression, deltaNp63 expression, EGFR expression, EFGR copy numbers, global gene expression, and histopathologic features in OPLs from the patients obtained at either baseline or at 3 months follow-up. Potential roles of these factors in progression of OPLs and oral cancer risk were evaluated. Results suggest that these alterations may outperform previously reported biomarkers and clinicopathologic parameters in stratifying oral cancer risk of patients with OPLs. Development of oral cancer risk assessment strategies faces a number of challenges. Etiologies of OPLs and oral cancer may change over time, such as the emerging evidence of HPV infection in orapharyngeal cancers. Populations tested in many of our studies may be biased and not representative of the general population. Therefore, results may be difficult to validate in independent populations. We lack of systematic research networks at the national level to facilitate oral cancer risk assessment, early detection, and prevention. Efforts are lacking to integrate oral cancer risk assessment and chemoprevention programs. Some progresses and thoughts towards these limitations will be discussed. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN14-04.

Retrospective study of the progression of oral premalignant lesions to squamous cell carcinoma: a South Wales experience.

The premalignant potential of leukoplakia is controversial. This study is intended to determine whether chronic oral "white lesions" demonstrated premalignant potential by investigating the population presented to the University Hospital of Wales in Cardiff between January 1996 and June 2000. Patient demographics are presented. During this period, 510 biopsies were performed on suspicious white lesions and 311 cases of oral cancer were diagnosed. During the same period, 11 of the 311 patients developed malignancy despite initial benign diagnosis. Thirty-four of the 510 biopsies were initially diagnosed as oral lichen planus (OLP), whereas 3 of the previously mentioned 11 were diagnosed as OLP and later developed squamous cell carcinoma. This represents an incidence of detected premalignancy in 3.5% of oral cancer cases. On re-examination of the initial biopsy specimens, these 3 cases met criteria for OLP in some areas, whereas the others appeared hyperkeratotic. These specimens may represent a subtype of premalignant chronic white lesions rather than exemplify the premalignant potential of OLP. Further direction of research will be discussed.

Loss of heterozygosity: a potential tool in management of oral premalignant lesions?

Oral leukoplakia, a heterogeneous group of lesions, demonstrates a varying degree of risk for cancer progression. Histology (presence and degree of dysplasia), the current gold standard for assessing this risk, is reasonably effective in judging the malignant risk of high-grade pre-invasive lesions. It is, however, a poor predictor for lesions without dysplasia, or with minimal dysplasia, as only a few of these lesions will progress to cancer. This poses an enormous dilemma for clinicians as to whether these lesions should be aggressively treated or not. Recent studies show that loss of specific chromosomal regions (loss of heterozygosity, LOH) that contain known or presumptive tumor suppressor genes is an early predictor of subsequent progression of oral premalignant lesions. Incorporation of LOH findings into staging of oral premalignancy could improve our ability to identify and manage high-risk premalignant lesions, particularly those with relatively benign histology but high-risk genetic changes (high-risk LOH pattern).

Expression of p16 and CDK4 in oral premalignant lesions and oral squamous cell carcinomas: a semi-quantitative immunohistochemical study.

The protein, p16, the product of cyclin-dependent kinase number 2 (CDKN2) gene, is one of the negative regulators of the cell cycle. CDK4, encoded by CDK4 gene, is the substrate of p16. We investigated the expression of p16 and CDK4 and their role in the progression of oral premalignant lesions (OPLs) and oral squamous cell carcinomas (OSCCs) in a Chinese cohort. A total of 74 samples were obtained from patients with hyperkeratosis (10), OPLs [30; mild (10), moderate (10) and severe (10) dysplastic lesions], OSCCs (15; 8 non-metastatic, 7 metastatic), and normal oral tissues (10), together with local lymph nodes (9) of patients with metastatic OSCCs. A labelled streptavidin biotin (LSAB) immunohistochemistry assay was performed and a semi-quantitative method was used to evaluate the staining intensity. The staining patterns of p16 and CDK4 were similar in all tissues and were located in both the nuclei and the cytoplasm. However, the antigen distribution characteristics and the degree of expression of both p16 and CDK4 varied at different developmental stages of the lesions. Hyperkeratotic and dysplastic lesions tended to display a progressively increasing degree of p16- and CDK4-positive nuclei as compared with normal tissues. Also, positive staining cytoplasm was highly evident in OSCCs with a very low nuclear staining (P<0.05). Forty-six of 56-, p16-positive cases were CDK4-positive, while only 6 were CDK4-positive but p16-negative, implying a high correlation between these parameters (r=0.813, P<0.001). This study indicates that the expression of p16 and CDK4 is intimately involved in the development of OPLs and OSCCs and the likely existence of a positive feedback loop between p16 and CDK4.